Lsm780 zen confocal microscope

Fluorescein isothiocyanate-labeled formaldehyde-denatured serum albumin (FITC-FSA; Sigma-Aldrich) was diluted to 10 μg/ml in endothelial medium and added to HSEC monolayers grown in rat-tail collagen-coated 0.4 channel μ-Slides VI (Ibidi). After 15 min, the channels were washed three times in PBS, the cells were fixed with 4% paraformaldehyde solution for 10 min, washed with PBS, and DAPI stained to identify nuclei, and FITC positivity was analyzed using a Carl Zeiss LSM780 Zen Confocal microscope (Carl Zeiss) and LSM software. Control cells were incubated with the same medium without the addition of FITC-FSA. Purity was assessed as the percentage of FITC-positive plated cells taken from six random fields.

Tissue sections from explant livers from alcoholic liver disease, primary sclerosing cholangitis, primary biliary cholangitis, and samples of normal liver from resections were initially incubated with blocking antibody to CD151 (IIG5a; AbD Serotec; 10 μg/ml) or control antibody. The sections were then overlaid with PBLs that had been prelabelled with cell tracker green (ThermoFisher Scientific); PBLs were resuspended at 1 × 10⁶/ml, and 100-μl aliquots were added to each section for 30 min at room temperature to adhere in static conditions. The nonadherent cells were removed, and the remaining adherent cells were fixed with acetone. The sections were then stained with DAPI and imaged on a Carl Zeiss LSM780 Zen Confocal microscope (Carl Zeiss), and lymphocytes present in a minimum of 10 representative high-power fields per section were counted.

For immunofluorescent staining of HSECs, cells were grown to confluence on rat tail collagen-coated eight-well glass-bottom μ-slides (Ibidi), stimulated (see Stimulation of HSECs) and subsequently fixed in 100% methanol. The cells were then blocked and permeabilized with a solution of PBS with 10% goat serum, 2× casein, and 0.1% Triton-X 100 (Sigma-Aldrich) for 20 min at room temperature. Primary antibodies, diluted in blocking buffer, were then added and incubated for 60 min, followed by isotype-specific fluorescence-conjugated goat anti-mouse secondary antibodies for 30 min. Finally, 300 nM DAPI was added to each chamber for 2 min before the mounting in aqueous Fluorescent Mounting Medium (Dako). All immunofluorescent staining was imaged on a Carl Zeiss LSM780 Zen Confocal microscope (Carl Zeiss) and analyzed by LSM software.